Pseudomonas aeruginosa is one of the most serious opportunistic Gram-negative bacterial pathogens, and is resistant to an alarming number of antibiotics. The bacterium causes problems in intensive care settings due to its propensity to colonize medical devices, and is a major cause of morbidity and mortality in people with cystic fibrosis. P. aeruginosa is also a serious problem in wound care, especially the chronic wounds of individuals that suffer from diabetes mellitus, or other chronic conditions affecting the circulatory and immune systems, and those with major burn injuries. Bacteria that colonize the wound grow in dense matrix-embedded communities or biofilms. Biofilms are a major contributing factor to lack of healing, as the matrix-embedded bacteria are recalcitrant to antibiotics and immunity, rendering them extremely challenging and costly to treat. Indeed, it is estimated that in the US alone $25 billion dollars are spent annually on the treatment of chronic wounds. The ineffectiveness of currently available standard of care treatments to eradicate biofilms, has led us to develop a series of therapeutic enzymes - carboclippers - that explicitly target and degrade the exopolysaccharide component of the biofilm matrix. Preliminary studies have focused on the development of enzymes that target the Pel and Psl polysaccharides of the P. aeruginosa biofilms prevalent in chronic wounds. The enzymes are effective at low nanomolar concentrations and can both prevent in vitro biofilm formation as well as rapidly disrupt existing biofilms. Using a multidisciplinary approach encompassing biochemical, cell biological, microbiological and animal studies, we seek to determine whether disrupting the biofilm matrix using specific enzyme treatments will potentiate existing antibiotics and the immune response, and hence promote wound healing. In the R21 phase, we will establish the effectiveness of the enzymes as novel therapeutics and evaluate their cytotoxicity. With quantifiable transition milestones in place that evaluate the enzymes abilities to reduce biofilm biomass, potentiate antimicrobials, improve neutrophil-mediate killing, while having no effect on mammalian cells, the R33 phase will develop a formulation for the enzymes that is compatible with the wound environment and determine the effectiveness of the enzymes in our animal model of chronic wound infection. The results of these studies will establish proof-of-concept that enzyme biofilm disruptors in combination with antimicrobials and the innate immune response improve wound healing. The results of our studies will position us for the required pharmacokinetic studies that are a prelude to clinical trials. Importantly, not only can our methodology be developed further for the treatment of other chronic P. aeruginosa infections, but the approach of using enzyme biofilm disruptors that hydrolyze polysaccharides is also applicable to biofilm infections caused by other pathogenic bacteria of relevance to this RFA including but not limited to Acinetobacter baumanii or pathogenic Escherichia coli.